Background: Mutations in the p53 tumor suppressor gene have been identified in breast and many other carcinomas. It is not clear, however, when these mutations occur during breast carcinogenesis. Overexpression of 53 protein has been reported in some ductal carcinoma in situ (DCIS) lesions. To further study the overexpression of p53 in DCIS of the breast and correlate these findings with changes at the molecular level, we performed p53 immunostaining and direct sequencing in noncomedo and comedo DCIS.
Experimental design: Archival blocks were obtained on cases of noncomedo DCIS, (including encysted, noninvasive papillary carcinoma) and comedo DCIS. Immunohistochemical staining with the p53 antibody DO7 was performed on all cases. Polymerase chain reaction (PCR) amplification of exons 5, 6, and 7 of the p53 gene was performed and the PCR products were directly sequenced. Four comedo DCIS cases that were p53 immunopositive were further screened for p53 mutations by PCR/single strand conformation polymorphism in exons 8 and 9 of the p53 gene. One of these cases showing a mobility shift was directly sequenced.
Results: We examined 39 lesions including comedo DCIS (N = 12) and noncomedo DCIS (N = 27), the latter including 17 encysted noninvasive papillary carcinomas. Immunostaining with DO7 was positive in 4 of 12 comedo DCIS lesions (33%) while all noncomedo lesions including encysted noninvasive papillary carcinomas were negative. Direct sequencing of PCR products confirmed wild-type DNA in exons 5 and 6 in all noninvasive papillary carcinomas, 3 randomly selected noncomedo DCIS lesions, and 4 p53 antibody-positive comedo DCIS lesions. In these latter 4 cases, wild-type DNA sequences were preserved in exon 7 for all cases. A single case had a conformational shift in exon 8 within the four cases screened in exons 8 and 9. Direct DNA sequencing of this exon revealed a G to A point mutation resulting in an arginine-to-histidine substitution at codon 273 of the protein.
Conclusions: Our results suggest that mutant p53 protein accumulation in preinvasive lesions is limited to comedo (high grade) DCIS and that p53 positivity by immunohistochemistry does not correlate in all cases with specific p53 mutations in exons 5 to 9, the most highly conserved regions of this gene.